The Vondriska lab is supported by grants from the National Heart, Lung and Blood Institute of the NIH and the Laubisch Endowment at UCLA. EM is recipient of the Jennifer S. Buchwald Graduate Fellowship in Physiology at UCLA; HC is the recipient of an American Heart Association Pre-doctoral Fellowship; MP is the recipient of an NIH Ruth Kirschstein Post-doctoral Fellowship; and SF is the recipient of an NIH K99 Award.

The experimental workflow contains seven major steps (Figure 1). For any work involving samples that will be run on the mass spectrometer, the experimenter should wear a lab coat, gloves and hair net and take care to avoid contamination from dust and personal shedding of keratin.
1. Heart Homogenization and Nuclear Isolation
Mouse hearts are homogenized and an intact nuclei pellet is isolated (Figure 2).
<ol>
 <li>Sacrifice adult mouse, excise the heart, rinse in ice-cold PBS, and homogenize on ice in glass dounce (we prefer the Wheaton Tissue Grinder from Fisher, #08-414-13A, but other methods may work equally well) containing 2 ml of lysis buffer (a hypotonic solution which differentially lyses the cell membrane over the organelles including the nucleus) (10 mM Tris pH 7.5, 15 mM NaCl, and 0.15% v/v Nonidet P-40 [NP-40] in deionized water plus protease and phosphatase inhibitor mixture: 10 mM sodium butyrate, 0.1 mM phenylmethylsulfonyl fluoride [PMSF], 0.2 mM Na3VO4, 0.1 mM NaF and 1 Roche protease pellet/10 ml - lysis buffer can be stored for up to one week at -20 &deg;C). (Note: We do not find it necessary to perfuse the hearts with PBS, as our mass spectrometry data and GO analysis identify the proteins as predominately cardiomyocyte [p-value 3.8E-22] in origin as opposed to blood contamination [p value 5.0E-2].)</li>
 <li>Pour lysate through 100 &mu;m strainer and collect flow through in 1.5 ml centrifuge tube. At this point, unless specified, sample should be kept on ice.</li>
 <li>Centrifuge at 4,000 rpm for 5 min at 4 &deg;C.</li>
 <li>Remove supernatant. (This is the cytosol, and should be stored at -80 &deg;C. It contains the lysed mitochondria.) Resuspend pellet in 200 &mu;l lysis buffer by triturating. (This is the crude nuclear pellet.)</li>
 <li>Fill 1.5 ml centrifuge tube with 1 ml of sucrose buffer (24% sucrose weight/volume, 10 mM Tris pH 7.5, and 15 mM NaCl in deionized water with protease/phosphatase inhibitor mixture - sucrose buffer should be made fresh on the day of use). Gently layer the resuspended pellet on top of the sucrose pad and centrifuge at 5,000 rpm for 10 min at 4 &deg;C.</li>
 <li>Remove the thin film on top as well as the sucrose pad (which contain membrane). Rinse the remaining pellet with 200 &mu;l ice-cold PBS/EDTA (1x PBS with 1 mM EDTA). (This is the nuclei pellet and can be solubilized or fixed for use in western blotting or electron microscopy [See steps 4.1 and 4.2] to quantify enrichment.)</li>
</ol>
2. Nucleoplasm and Detergent-extracted Chromatin Fractionation
The crude nuclei pellet is separated into a nucleoplasm and detergent-extracted chromatin fraction, containing proteins loosely associated with the DNA.
<ol>
 <li>Triturate pellet from step 1.6 in 200 &mu;l detergent extraction buffer (20 mM HEPES pH 7.6, 7.5 mM MgCl2. 0.2 mM EDTA, 30 mM NaCl, 1 M Urea, 1% NP-40 in deionized water with protease/phosphatase inhibitor mixture - detergent extraction buffer can be stored for up to one week at -20 &deg;C).</li>
 <li>Vortex sample 2 times, 10 sec each. Place on ice 10 min.</li>
 <li>Centrifuge at 13,000 rpm for 5 min at 4 &deg;C.</li>
 <li>Remove supernatant. (This is the nucleoplasm and should be stored at -80 &deg;C). Rinse pellet with ice-cold PBS/EDTA. (This is the chromatin pellet.)</li>
 <li>Triturate pellet in 300 &mu;l Tris, SDS, EDTA buffer (50 mM Tris pH 7.4, 10 mM EDTA, 1% SDS in deionized water with protease/phosphatase inhibitor mixture - Tris, SDS, EDTA buffer can be kept for up to one week at -20 &deg;C).</li>
 <li>Sonicate 3-6 times for 10 sec each to break up DNA. Keep sample on ice between sonications.</li>
 <li>Centrifuge at 13,000 rpm for 5 min at 4 &deg;C. Keep the supernatant. (Supernatant is the detergent-extracted chromatin protein fraction and should be kept at -80 &deg;C). The remaining pellet should be small and can be discarded.</li>
 <li>If using isolated myocytes instead of the whole heart: Isolate neonatal rat ventricular myocytes from rat pups one day after birth using enzymatic digestion, followed by culture in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS), 1% insulin-transferrin-sodium selenite (ITS), and 1% penicillin. After 24 hr, transfer to serum-free media (same as above, but lacking FBS). Harvest cells in lysis buffer (listed in 1.1), and begin nuclear fractionation at step 1.3.</li>
</ol>
3. Acid-extraction Fractionation - DNA-bound Protein Enrichment
A separate fractionation enriches for proteins tightly bound to the DNA, including histones.
<ol>
 <li>Repeat steps 1.1-2.4.</li>
 <li>Triturate the chromatin pellet in 400 &mu;l of 0.4 N sulfuric acid. Vortex to remove clumps.</li>
 <li>Incubate at 4 &deg;C for 30 min or overnight while rotating.</li>
 <li>Centrifuge at 16,000 x g for 10 min at 4 &deg;C to remove nuclear debris.</li>
 <li>Transfer supernatant to a fresh tube.</li>
 <li>Add 132 &mu;l of trichloroacetic acid drop-wise to the supernatant. Invert several times. Incubate on ice for 30 min.</li>
 <li>Centrifuge at 16,000 x g for 10 min at 4 &deg;C.</li>
 <li>Discard supernatant. Gently rinse pellet with ice-cold acetone. (This is the histone pellet.)</li>
 <li>Centrifuge at 16,000 x g for 10 min at 4 &deg;C.</li>
 <li>Repeat wash, steps 3.8-3.9.</li>
 <li>Air-dry pellet.</li>
 <li>Resuspend the pellet in 100 &mu;l of Tris, SDS, EDTA buffer. Set pH to 8 by adding 1 M Tris. (Use a 1 M Tris stock that is not pH-adjusted.)</li>
 <li>Sonicate in water bath for 15 min. Prevent overheating by adding ice to water bath. (This is the acid-extracted fraction and should be stored at -80 &deg;C.)</li>
</ol>
4. Purity Check
Western blots and electron microscopy confirm successful enrichment of nuclei and depletion of other organelles. To see typical results for all of the following quality control assays, please see our previous publication.18
<ol>
 <li>Perform Western blot analysis. Probe membrane containing each fraction for histone H2A or nucleoporin p62 (as nuclear markers to verify enrichment), and for adenine nucleotide transporter, BiP and tubulin (as a mitochondrial marker, endoplasmic reticulum marker, and cytoskeletal marker respectively to verify purity). To verify successful subfractionation of the nuclei, probe for histone H3 or fibrillarin (detergent and acid-extracted chromatin and intact nuclei) and SNRP70 or E2F (nucleoplasm). Probe for retinoblastoma or hypoxia inducible factor-1 (enriched in detergent-extracted chromatin over acid-extracted fraction). Additional control samples of HeLa cell lysate and whole heart lysate can also be run in the same gel: Prepare HeLa cell lysate control by adding Tris, SDS, EDTA buffer to culture dish and using a cell scraper to collect sample. Sonicate and centrifuge sample as in steps 2.6-2.7. Prepare whole heart lysate control by homogenizing the heart in 2 ml buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate in deionized water with protease and phosphatase inhibitor mixture). Sonicate and centrifuge as in steps 2.6-2.7. See step 5 for preparing protein samples for SDS-PAGE.</li>
 <li>Electron Microscopy: Resuspend the nuclei pellet from step 1.6 in lysis buffer containing 2% gluteraldehyde and fix sample at 4 &deg;C. Rinse samples in osmic acid, dehydrate, and embed in epoxy resin. Cut 70 nm slices using a Reichert Ultracut ultramicrotome. Stain samples in uranyl acetate and then lead and image using a JEOL 100CX Transmission Electron Microscope. Quantify enrichment by measuring the area of intact nuclei versus total area of material imaged. We find 60-80% of nuclei to be intact.14</li>
</ol>
Important considerations for enriching versus purifying nuclei. This protocol was developed to enable proteomic analyses of cardiac chromatin for the purpose of studying global gene regulation processes during disease. A major component of designing whole-proteome mass spectrometry experiments is the issue of dynamic range and being able to identify and characterize low-abundance proteins. In addition to the primary goal of providing information on nuclear and chromatin-specific biology, enriching for the nuclear proteome, increases the likeliness of detecting these low-abundance proteins, as does further subfractionation of the nuclei. However, as discussed, the adult cardiomyocyte has a dense cytoskeletal component connected to the nuclear membrane. We do not believe that we have completely purified the nuclei from these other cellular components. However, by enriching only for the nuclei, we have obtained an acceptable threshold to enable the types of analyses described.
Specifically, we have used EM to assess the purity of our crude nuclei pellet and found 60-80% of the fraction to be intact nuclei, roughly 10% mitochondria, and the rest debris. Additionally, we know from our previous study on the intact nuclei population, that while many myofilaments are not enriched by this protocol (including tubulin and actin as measured by Western) certain proteins (calsarcin-1) are enriched, suggesting a true biological population of these proteins in the cardiac nuclei.19
Additionally, we compared the proteins we found to be present in the acid-extracted chromatin fraction, to the predicted roles of these proteins by gene ontology. Importantly, this gene ontology analysis does not take into account the relative abundance of the different proteins (as does our Western blot data) but rather counts all proteins identified (enriched or not) as equal when identifying common pathways and cellular compartments. Analysis of these pathways and cellular compartments can be found in our previous publications.18,20 Most importantly, the success of the enrichment in the acid-extracted chromatin fraction allowed us to identify the presence of 54 histone variants in the adult mouse myocyte,18 many of which relied on one unique peptide for identification, and thus would likely not have been detectable without this enrichment protocol given the enormous complexity of the total cardiomyocyte proteome. Of the 1,048 proteins we identified from the cardiac nuclei, 56 of them (5.3%) were annotated by GO analysis to be part of the nucleosome (one component of the nucleus of interest). Another study looking at the whole heart, identified 6,180 proteins, of which only 11 proteins (0.18%) were annotated to be part of the nucleosome21. This further illustrates the strength of our protocol to meaningfully enrich for nuclear proteins.
5. Protein Gel and Enzymatic Protein Digest
Proteins are separated by one dimensional SDS-PAGE and trypsin digested to be run on the mass spectrometer.
<ol>
 <li>Thaw samples on ice and determine protein concentration for each sample using the bicinchoninic acid (BCA) protein assay.</li>
 <li>Dilute samples to a known concentration using 5x Laemmli buffer, boil for 10 min and store at -20 &deg;C for one-dimensional SDS-PAGE. Dilute samples to a known concentration using urea extraction buffer and store at -20 &deg;C for two-dimensional gels.</li>
 <li>Run gel of choice loading equal amount of protein in each lane. Protein can be transferred to a nitrocellulose membrane to be used for Western blotting (as with controls in step 4.1) or bands can be cut for mass spectrometry analysis; see following steps.</li>
 <li>Remove gel from apparatus using deionized water to transfer it to a clean container. Cover gel in Oriole stain, and wrap container in aluminum foil to block light. Allow gel to incubate at room temperature on a shaker for at least 90 min.</li>
 <li>Image Oriole-stained gel (Figure 3) using UV light, and mark where bands will be cut on the image. We cut each lane into approximately 25 2-mm bands for studies measuring the total proteome.</li>
 <li>Place gel onto a clean surface. Use only materials that have been kept sealed or are sprayed with ethanol to prevent keratin contamination. Cut out each band, and then further slice it into 3 equal pieces. Place all three pieces of each band together into its own labeled 1.5 ml tube. Gel pieces can be stored at -20 &deg;C for several months.</li>
 <li>Prepare gel plugs for enzymatic digestion. Digest gel pieces using trypsin at 37 &deg;C overnight. For detailed gel sample protocol see our previous publication.22 You can digest low molecular weight bands with chymotrypsin in lieu of trypsin, to eliminate trypsin's extensive cleavage of histone tails.</li>
</ol>
6. Mass Spectrometry and Data Analysis
Samples are separated on an LC and analyzed by MS/MS. The spectra are searched against a protein database for protein identification.
<ol>
 <li>Run 10 &mu;l of each sample through LC/MS/MS. We use a nano-flow Eskigent LC with a 75 &mu;m reverse phase column. Use an LC run optimized for a range of protein and peptides. Employ a linear gradient from mobile phase A (0.1%formic acid, 2%acetonitrile [ACN] in water) to mobile phase B (0.1%formic acid, 20%water in ACN): 60 min from 5% mobile phase B to 50% B, then 15 min from 50% B to 95% B and finally 10 min at constant 95% B. Use a flow rate of 200 nl/min. Acquire mass spectrometry data in a data-dependent mode. We use a Thermo Orbitrap that fragments the top 6 most abundant parent ions.</li>
 <li>Repeat runs for at least three biological and two technical replicates. (Recommended)</li>
 <li>Use software (commercially available options include BioWorks and Xcalibur; publically available options include PROWL, X!Tandem, SpectraST) to search spectra against the Uniprot database of choice via a search algorithm (such as SEQUEST or MASCOT).</li>
 <li>Consider modifying search parameters to allow for cysteine carbamidomethylation and methionine oxidation, two common modifications created during the sample processing.</li>
 <li>Calculate a false positive rate using reverse database searching.</li>
 <li>Filter protein identifications to only accept matches of a threshold confidence. We recommend the following parameters to start with: Xcorr &gt;3 (+2), &gt;4 (+3), &gt;5 (+4); deltaCN &gt;0.1, consensus score &ge;20, mass tolerance 2 Da for parent ion, mass tolerance of 0.5 Da for product ion, at least 2 unique peptides per protein and no more than 3 missed cleavages.</li>
</ol>
7. Label-free Quantitation
Determine the relative abundance of proteins using label-free quantitation (Figure 4).
<ol>
 <li>A number of software programs are publically or commercially available for label-free quantitation of mass spectrometry data including Census (Prof. Yates' group),23 Elucidator (Microsoft),24 SIEVE (Thermo Scientific),25 Scaffold (Proteome Software).26 These programs aim to correlate the mass spectrometric signal of intact peptides or the number of peptide sequencing events with relative protein quantities between two or more states.</li>
 <li>While each program incorporates a similar analysis pipeline, some programs are limited to the types of specific analysis that can be performed on the data. Initially, data from different runs is aligned, signal intensity is normalized and peptide peaks are selected for analysis.</li>
 <li>The two most common methods are quantification based on spectral counting or LC-MS peak area. Abundance ratios are calculated to determine changes in peptide abundance between different groups.</li>
 <li>These programs can be interfaced with proteomic search algorithms (Mascot, Sequest, X!Tandem) to correlate quantification information to protein identification.</li>
 <li>To ensure accuracy and reproducibility of the data it is crucial to incorporate both biological (different experimental samples) and technical (running the same sample on the mass spectrometer multiple times) replicates of mass spec data.</li>
 <li>Variation of peptide abundance in and between experimental conditions can be assessed by ANOVA and plotted via PCA (Figure 5).</li>
</ol>
Important considerations for label-free quantitation. When performing label-free quantitation, specific attention must be given to ensure consistent sample preparation, digestion time and LC-MS/MS conditions as each sample must be processed and analyzed separately. In contrast to metabolic labeling approaches, comparisons in label-free experiments are made on data from distinct mass spectrometry runs (because the lack of labeling obviates the possibility of running them together), thereby necessitating high reproducibility in all aspects of sample analysis (i.e. of sample prep, LC and MS steps) and use of high mass accuracy mass spectrometers.
To account for slight changes in sample preparation and analysis a known standard may be added to each sample to assist in normalization of data. Additionally, most software programs allow normalization of signal intensities (e.g. by adjusting to background noise or a known abundant analyte) after data acquisition to account for differences in injection, ionization and fragmentation. Alignment algorithms exist in most of the above-referenced software programs that assist in correcting differences in peptide elution profiles. The use of biological and technical replicates is essential in this type of study, as it allows statistical analyses to confirm the reproducibility and consistency of any observed changes in protein abundance.

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J. 77, 631-636 (1960).</li><li>Rodriguez-Collazo, P., Leuba, S. H., Zlatanova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+methods+for+purification+of+histones+from+cultured+mammalian+cells+with+the+preservation+of+their+native+modifications.">Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.</a> Nucleic Acids Res. 37, e81 (2009).</li><li>Kuehl, L., Salmond, B., Tran, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentrations+of+high-mobility-group+proteins+in+the+nucleus+and+cytoplasm+of+several+rat+tissues.">Concentrations of high-mobility-group proteins in the nucleus and cytoplasm of several rat tissues.</a> J. Cell Biol. 99, 648-654 (1984).</li><li>Gronow, M., Griffiths, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+and+separation+of+the+non-histone+proteins+of+rat+liver+nuclei.">Rapid isolation and separation of the non-histone proteins of rat liver nuclei.</a> FEBS Lett. 15, 340-344 (1971).</li><li>Mischke, B. G., Ward, O. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+tissue+specificity+of+chromatin-associated+proteins+in+Vicia+faba.">Isolation and tissue specificity of chromatin-associated proteins in Vicia faba.</a> Can J. Biochem. 53, 91-95 (1975).</li><li>Andersen, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+proteomic+analysis+of+the+human+nucleolus.">Directed proteomic analysis of the human nucleolus.</a> Curr. Biol. 12, 1-11 (2002).</li><li>Zhao, Y., Jensen, O. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification-specific+proteomics:+strategies+for+characterization+of+post-translational+modifications+using+enrichment+techniques.">Modification-specific proteomics: strategies for characterization of post-translational modifications using enrichment techniques.</a> Proteomics. 9, 4632-4641 (2009).</li><li>Ahrne, E., Muller, M., Lisacek, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unrestricted+identification+of+modified+proteins+using+MS/MS.">Unrestricted identification of modified proteins using MS/MS.</a> Proteomics. 10, 671-686 (2010).</li><li>Young, N. L., Plazas-Mayorca, M. D., Garcia, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems-wide+proteomic+characterization+of+combinatorial+post-translational+modification+patterns.">Systems-wide proteomic characterization of combinatorial post-translational modification patterns.</a> Expert Rev. Proteomics. 7, 79-92 (2010).</li><li>Guthals, A., Bandeira, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+identification+by+tandem+mass+spectrometry+with+alternate+fragmentation+modes.">Peptide identification by tandem mass spectrometry with alternate fragmentation modes.</a> Mol. Cell Proteomics. , (2012).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protease+for+'middle-down'+proteomics.">A protease for 'middle-down' proteomics.</a> Nat. Methods. , (2012).</li><li>Tran, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+intact+protein+isoforms+in+discovery+mode+using+top-down+proteomics.">Mapping intact protein isoforms in discovery mode using top-down proteomics.</a> Nature. 480, 254-258 (2011).</li></ol>

No conflicts of interest declared.

Two main methods for nuclear isolation have been reviewed previously:27 one is the Behrens technique of homogenizing lyophilized tissue in a non-aqueous solvent and the second, a modification of which we use here, of homogenizing tissue in an aqueous sucrose/salt solution followed by differential or density-gradient centrifugation.
Subfractionation of the nuclei by acid extraction on tissue samples is an important tool for studying chromatin which has been used since 1960,28</s...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Dulbeco Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965</td>
		</tr>
		<tr>
			<td>Protease pellet</td>
			<td>Roche</td>
			<td>04 693 159 001</td>
		</tr>
		<tr>
			<td>100 &mu;m strainer</td>
			<td>BD Falcon</td>
			<td>352360</td>
		</tr>
		<tr>
			<td>Ultracut ultramicrotome</td>
			<td>Reichert</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100CX Transmission Electron 
			Microscope</td>
			<td>JEOL USA, Inc.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oriole</td>
			<td>BioRad</td>
			<td>161-0496</td>
		</tr>
		<tr>
			<td>Histone H2A antibody</td>
			<td>Santa Cruz</td>
			<td>sc-8648</td>
		</tr>
		<tr>
			<td>Nucleoporin p62 antibody</td>
			<td>BD Biosciences</td>
			<td>610498</td>
		</tr>
		<tr>
			<td>Adenine nucleotide transporter antibody</td>
			<td>Santa Cruz</td>
			<td>sc-9299</td>
		</tr>
		<tr>
			<td>BiP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-1050</td>
		</tr>
		<tr>
			<td>Tubulin antibody</td>
			<td>Sigma</td>
			<td>T1568</td>
		</tr>
		<tr>
			<td>Histone H3 antibody</td>
			<td>Abcam</td>
			<td>ab1791</td>
		</tr>
		<tr>
			<td>Fibrillarin antibody</td>
			<td>Cell Signaling</td>
			<td>C12C3</td>
		</tr>
		<tr>
			<td>SNRP70 antibody</td>
			<td>Abcam</td>
			<td>ab51266</td>
		</tr>
		<tr>
			<td>E2F-1 antibody</td>
			<td>Thermo Fisher</td>
			<td>MS-879</td>
		</tr>
		<tr>
			<td>Retinoblastoma antibody</td>
			<td>BD Biosciences</td>
			<td>554136</td>
		</tr>
		<tr>
			<td>Hypoxia inducible factor-1 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB100-469</td>
		</tr>
		<tr>
			<td>BCA protein assay</td>
			<td>Thermo Scientific</td>
			<td>23227</td>
		</tr>
		<tr>
			<td>Reverse phase column</td>
			<td>New Objective</td>
			<td>PFC7515-B14-10</td>
		</tr>
		<tr>
			<td>BioWorks Browser</td>
			<td>Thermo Scientific</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

quantitative analysis of chromatin proteomes in disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Figure 4 highlights the utility of this form of relative quantification. Shown in the left panel are the individual monoisotopic peptide peaks (overlaid from different mice), which have been designated as belonging to the protein HMGB1 (identified via database search). Each peak, essentially an extracted ion chromatograph for the given peptide, comes from a different mouse. The groups represent three different physiological states: basal, cardiac hypertrophy, and heart failure, with three biological repl...

Watch this Scientific Journal Video about Quantitative Analysis of Chromatin Proteomes in Disease at JoVE.com

Quantitative Analysis of Chromatin Proteomes in Disease

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

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13.0K Views.  David Geffen School of Medicine at UCLA. Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

Video: Quantitative Analysis of Chromatin Proteomes in Disease

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer's Disease and Mild Cognitive Impairment

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior Cingulate Cortex (PCC) is a potential imaging marker for monitoring the progression of AD. Previous studies did not focus on the functional connectivity between the PCC and nodes in regions outside the DMN, but our study is an effort to explore these overlooked functional connections. For collecting data, we used functional Magnetic Resonance Imaging (fMRI) and Granger Causality Analysis (GCA). fMRI provides a non-invasive method for studying the dynamic interactions between the different brain regions. GCA is a statistical hypothesis test for determining whether one-time series is useful in forecasting another. In simple terms, it is judged by comparing the &#34;Known all the information on the last moment, the distribution of the probability of X at this time&#34; and the &#34;Known all the information on the last moment except Y, the distribution of the probability of X at this time&#34;, to determine whether there is a causal relationship between Y and X. This definition is based on the complete information source and stationary chronological sequence. The main step of this analysis is to use X and Y to establish the regression equation and draw a causal relationship by a hypothetical test. Since GCA can measure causal effects, we used it to investigate the anisotropy of the functional connectivity and explore the hub function of the PCC. Here, we screened 116 participants for MRI scanning, and after preprocessing the data obtained from neuroimaging, we used GCA to derive the causal relationship of each node. Finally, we concluded that the directed connection is significantly different between the Mild Cognitive Impairment (MCI) and AD groups, both from the PCC to the whole brain and from the whole brain to the PCC.

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer&#039;s Disease and Mild Cognitive Impairment

Based on resting-state functional magnetic resonance imaging with Granger causality analysis, we investigated the alterations in the directed functional connectivity between the posterior cingulate cortex and whole brain in patients with Alzheimer&#39;s Disease (AD), patients with Mild Cognitive Impairment (MCI), and healthy controls.

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior ...

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

&lt;em&gt;In Vivo&lt;/em&gt; Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&amp;#232;re&#039;s Disease Ears and Normal Hearing Ears

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&#8212;Application in Premanifest Huntington's Disease

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy begins many years prior to the motor onset (during the &#34;premanifest&#34; period), but the spatiotemporal pattern of regional atrophy across the brain has not been fully characterized. Here we demonstrate an online cloud-computing platform, &#34;MRICloud&#34;, which provides atlas-based whole-brain segmentation of T1-weighted images at multiple granularity levels, and thereby, enables us to access the regional features of brain anatomy. We then describe a regression model that detects statistically significant inflection points, at which regional brain atrophy starts to be noticeable, i.e. the &#34;change-point&#34;, with respect to a disease progression index. We used the CAG-age product (CAP) score to index the disease progression in HD patients. Change-point analysis of the volumetric measurements from the segmentation pipeline, therefore, provides important information of the order and pattern of structural atrophy across the brain. The paper illustrates the use of these techniques on T1-weighted MRI data of premanifest HD subjects from a large multicenter PREDICT-HD study. This design potentially has wide applications in a range of neurodegenerative diseases to investigate the dynamic changes of brain anatomy.

Whole-brain Segmentation and Change-point Analysis of Anatomical Brain MRI&amp;#8212;Application in Premanifest Huntington&#039;s Disease

This paper describes a statistical model for volumetric MRI data analysis, which identifies the &#34;change-point&#34; when brain atrophy begins in premanifest Huntington&#39;s disease. Whole-brain mapping of the change-points is achieved based on brain volumes obtained using an atlas-based segmentation pipeline of T1-weighted images.

Recent advances in MRI offer a variety of useful markers to identify neurodegenerative diseases. In Huntington&#39;s disease (HD), regional brain atrophy ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...